PROJECT SUMMARY Historically men have been diagnosed with alcohol use disorders (AUDs) at higher rates than women, but greater risk for adverse health consequences is observed in women who are problem drinkers. In recent years, the gap in diagnosis of AUDs between women and men has been closing. Despite this, minimal research has investigated factors mediating sex differences in relapse-related behavior. Men and women appear to be differentially sensitive to the ability of alcohol-paired cues or stress to drive alcohol craving or relapse-related behaviors, such that men are particularly sensitive to reward-paired cues, while women show escalated stress- induced craving. Animal models of relapse to alcohol seeking indicate that male rodents are similarly susceptible to cue-induced reinstatement, while females show elevations in stress-facilitation of reinstatement. The neural circuits and signaling systems that mediate these sex differences are only beginning to be understood. Nucleus accumbens (NAc) glutamate signaling is a critical regulator of reinstatement to ethanol seeking, and discrete glutamatergic projections play separable roles in stress- and cue-induced reinstatement. Further, pharmacological regulation of glutamate signaling can reduce reinstatement to ethanol seeking. Despite strong evidence that glutamate signaling within the NAc is critical for the regulation of reinstatement in males, sex differences in engagement of glutamatergic projections to the NAc or in expression of glutamate receptors and transporters following chronic alcohol exposure has not been extensively investigated. This R21 proposal will test the overarching hypothesis that sex differences in accumbens glutamate circuit function result in differential propensity toward stress- and cue-induced reinstatement. Specifically, we will assess innate and ethanol dependence-driven sex differences in neural function during cue- or stress-induced reinstatement. Aim 1 is designed to test the hypothesis that chronic alcohol exposure promotes reinstatement with sex-specific patterns. We expect that CIE facilitates cue-induced reinstatement in males, but stress-induced reinstatement in females. We will further use photometric assessment of calcium signaling in infralimbic, ventral hippocampus, and basolateral amygdalar glutamatergic projections to the NAc to determine if these circuits are engaged during cue- and stress-induced reinstatement in a sex- and ethanol dependence-driven manner. Aim 2 will investigate sex-specific effects of CIE on the expression of glutamate receptors and transporters in the NAc through the use of immunofluorescence and RNAscope in situ hybridization. The results of these experiments are expected to provide considerable information on the sex differences in neural circuits and environmental factors regulating reinstatement of ethanol seeking. We will also gain significant knowledge of sex differences in the impact of chronic alcohol exposure on glutamate signaling and relapse-related behavior. These findings are expected to enable development of novel drug targets for the reduction of relapse behavior as well as the establishment of targeted treatments strategies for men and women.